Investigating the requirement and regulation of vascular progenitors derived from the Second Heart Field in Pharyngeal Arch Artery development

咽弓动脉发育中第二心区来源血管祖细胞的需求和调节研究

• 批准号: 10153462
• 负责人: AnnJosette Ramirez
• 金额: $ 4.02万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10153462
• 关键词: Address; Affect; Aorta; Arteries; Attenuated; Blood Vessels; Branchial arch structure; Cardinal vein; Cell Count; Congenital Abnormality; Data; Defect; Development; DiGeorge Syndrome; Dorsal; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Ensure; Etiology; Financial compensation; Heart; Human; Hypoxia; Immunohistochemistry; In Situ Hybridization; KDR gene; Knock-out; Lead; Life; Live Birth; Lung; Maps; Mediating; Mus; Pathway interactions; Patients; Phenotype; Population; Process; Regulation; Reporter; Role; Source; Testing; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factors; aortic arch; conditional knockout; congenital heart disorder; disease phenotype; driving force; endothelial stem cell; insight; mouse model; mutant; notch protein; progenitor; recruit

PROJECT SUMMARY The improper formation or remodeling of the Pharyngeal Arch Arteries (PAAs) are among some of the most severe and life-threatening forms of congenital heart disease, such as those seen in patients with DiGeorge Syndrome. We have previously shown that the PAA endothelium originates from a subpopulation of mesodermal progenitors termed the Second Heart Field (SHF). In addition, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an RTK which is required for vascular development is highly expressed in these SHF progenitors as they contribute the PAAs. However, to what extent SHF-derived ECs are required for PAA formation, and the mechanisms which regulate the contribution of SHF progenitors to PAAs remains unclear. To address this, we developed a mouse model to conditionally ablate VEGFR2 from the SHF by crossing VEGFR2GFP/+;Isl1Cre/+ and VEGFR2f/f;Rosa26mTmG/mTmG, with the initial hypothesis that the loss of VEGFR2 from SHF vascular progenitors would lead to the absence of PAAs. Unexpectedly, we saw VEGFR2-positive endothelium in VEGFR2GFP/f;Isl1Cre/+ mutant embryos in which VEGFR2 is deleted in the SHF. However, pharyngeal endothelium in these mutants was hypoplastic and disorganized in comparison to control embryos. The use of the Cre reporter Rosa26mTmG demonstrated that these VEGFR2-positive ECs are not of SHF origin but instead, are from another progenitor source, suggesting a possible role for endothelial compensation. Interestingly, embryos heterozygous for VEGFR2 in the SHF lineage showed an impediment in SHF progenitor contribution, in addition to the disorganized and irregular phenotype seen in the knockout embryos, suggesting a possible haploinsufficiency of VEGFR2 in the SHF. Thus, we hypothesize that the SHF contains vascular progenitors essential for proper formation and remodeling of the PAAs. Further, we hypothesize that endothelial cell (EC) progenitors from pre-existing vasculature contributes to the PAAs when SHF contribution is attenuated. Here, we propose to test these hypotheses by addressing the following specific aims: 1) To determine the requirement and mechanisms regulating SHF-derived EC contribution in PAA development; 2) To identify the EC progenitor source(s) that contribute to PAA development when SHF-derived EC contribution is compromised; 3) To determine mechanism(s) that regulate the compensation of PAA ECs when SHF-derived EC contribution is compromised. We will utilize conditional knockout mouse models and immunohistochemistry to fate map and identify the requirement of SHF vascular progenitors in PAA development, and to identify the sources of the non- SHF-derived ECs in the PAAs of embryos null for VEGFR2 in the SHF. Furthermore, we will use a combination of immunohistochemistry and in situ hybridization to provide insight on the mechanisms regulating these vascular progenitors in PAA development. Elucidating sources and mechanisms that drive PAA formation will lead to a better understanding of CHD etiology.

项目摘要 咽弓动脉（PAA）的不适当形成或重塑是其中一些最重要的 严重和危及生命的先天性心脏病，如DiGeorge患者中所见 综合征我们之前已经证明，PAA内皮细胞起源于中胚层的一个亚群 称为第二心脏场（SHF）的祖细胞。此外，血管内皮生长因子受体2 血管内皮生长因子受体2（VEGFR 2），血管发育所需的RTK，在这些SHF祖细胞中高度表达， 他们贡献了PAA。然而，在何种程度上SHF衍生的EC是PAA形成所必需的， 调节SHF祖细胞对PAA的贡献的机制仍不清楚。为了解决这个问题，我们 开发了一种小鼠模型，通过将VEGFR 2GFP/+、Isl 1Cre/+和 VEGFR 2f/f; Rosa 26 mTmG/mTmG，最初的假设是SHF血管祖细胞中VEGFR 2的丢失 会导致PAA的缺失。出乎意料的是，我们看到VEGFR 2阳性的内皮细胞， VEGFR 2GFP/f;其中VEGFR 2在SHF中缺失的IsllCre/+突变胚胎。然而，咽部 与对照胚胎相比，这些突变体中的内皮发育不全且紊乱。使用 Cre报道基因Rosa 26 mTmG证明这些VEGFR 2阳性EC不是SHF来源的， 来自另一个祖细胞来源，提示内皮补偿的可能作用。有趣的是， SHF谱系中VEGFR 2杂合的胚胎显示SHF祖细胞贡献的障碍， 除了在敲除胚胎中观察到的无序和不规则的表型外，这表明可能的 SHF中VEGFR 2的单倍不足。因此，我们假设SHF含有血管祖细胞， 对于PAA的正确形成和重塑至关重要。此外，我们假设内皮细胞（EC） 当SHF贡献减弱时，来自预先存在的脉管系统的祖细胞对PAA有贡献。在这里， 我们建议通过解决以下具体目标来检验这些假设：1）确定要求 以及调控SHF衍生的EC在PAA发育中的作用的机制; 2）鉴定EC祖细胞 当SHF衍生EC贡献受损时，有助于PAA发展的来源; 3） 确定当SHF衍生的EC贡献是 暴露了我们将利用条件性基因敲除小鼠模型和免疫组织化学来绘制命运图， 确定PAA发育中SHF血管祖细胞的需求，并确定非SHF血管祖细胞的来源。 SHF中VEGFR 2无效的胚胎的PAA中的SHF衍生的EC。此外，我们将使用一个组合 免疫组织化学和原位杂交，以提供对调节这些血管的机制的见解， PAA发育中的祖细胞。阐明驱动PAA形成的来源和机制将导致 更好地了解CHD病因。